Head and neck cancer is one of the top five most common forms of malignancy. One of main problems in head and neck cancer treatment is local regional control and frequent recurrence. In this grant application, we propose to examine a hypothesis that may significantly improve the radiotherapy of H&N cancer. The long-term objective of this project is to characterize the status of the so-called master switches: hypoxia-inducible factor (HIF) genes, during radiotherapy of head and neck cancer and to exploit such knowledge for potential therapeutic gain. The hypothesis of our project is that HIF-1&2 genes play key roles in determining head and neck (H&N) cancer responses to radiotherapy, and that inhibiting HIF-1&2 activities can enhance the efficacy of H&N cancer radiotherapy. Our project is based on previous studies indicating that hypoxia and both of the HIF-1&2 factors play important roles in determining prognosis of head and neck cancer radiotherapy. In addition, it is based on our new data that indicated HIF factors were activated by radiotherapy independent of hypoxia through the generation of intratumoral nitric oxide, which can stabilize the alpha subunits of the HIF genes. We will conduct experiments to systematically characterize radiation-induced HIF-1 and HIF-2 gene activation in preclinical head and neck cancer models by use of novel molecular imaging and transgenic mouse approaches. Specifically, we will examine the roles of specific nitric oxide synthase genes in radiation-induced HIF-1&2 activation (Specific aim 1). In addition, we will attempt to decipher the molecular mechanism through which nitric oxide mediates radiation-induced HIF-1&2 activation in head and neck cancer radiotherapy (Specific aim 2). Finally, we will evaluate the relative importance of these two factors in head and neck cancer survival after radiotherapy (Specific Aim 3). The proposed studies should provide important insights into the biological mechanisms of radiation-induced activation of the HIF genes and evaluate the efficacy for inhibiting these two factors during head and neck cancer radiotherapy.